There are numerous methods of chromatography available today, such as ion exchange chromatography (IEX), hydrophobic interaction chromatography (HIC), reversed phase chromatography (RPC), affinity chromatography, gel filtration etc. The feature that distinguishes chromatography from most other physical and chemical methods of separation is that two mutually immiscible phases are brought into contact wherein one phase is stationary and the other mobile. The sample mixture, introduced into the mobile phase, undergoes a series of interactions i.e. partitions many times between the stationary and mobile phases as it is being carried through the system by the mobile phase. Interactions exploit differences in the physical or chemical properties of the components in the sample. These differences govern the rate of migration of the individual components under the influence of a mobile phase moving through the stationary phase. Separated components emerge in a certain order, depending on their interaction with the stationary phase. The least retained component elutes first, the most strongly retained material elutes last. Separation is obtained when one component is retarded sufficiently to prevent overlap with the zone of an adjacent solute as sample components elute from the column.
Chromatographic separation methods are useful e.g. for recovery of biomolecules, such as nucleic acids and proteins, and small organic molecules from liquids. In addition, chromatographic separation methods can also be used for recovery of liquids, in which case impurities such as organic or inorganic molecules are removed to result in a liquid of higher purity.
The matrix used in chromatography is composed of a carrier material, which is usually in the form of particles, monolithic matrices or the like. In some applications, such as RPC, the surface of the carrier itself will provide the interaction with the target molecules. In other applications, such as ion exchange or affinity chromatography, the carrier has been provided with ligands that comprise charged groups or affinity groups for interaction with target molecules. In either case, the carrier materials used can be classified as inorganic materials, such as silica, and organic material, including the groups of synthetic polymers, such as poly(divinylbenzene) (DVB) and poly(divinylbenzene/styrene), and native polymers, such as agarose.
Silica has been used for several years as chromatography beads. However, a general problem with silica is that it is susceptible to hydrolysis in basic conditions, and accordingly the conventional cleaning in place (cip) procedure using sodium hydroxide is not suitable in this case. For the same reason, silica matrices cannot be operated in chromatography under high pH conditions, which severely limits their use in a wide range of applications. More recently, alkyl-bonded silica particles have been suggested for reversed phase liquid chromatography. However, alkyl-bonded silica contains residual silanol groups or impurities, which in some cases results in peak tailing and a poor chromatographic performance in general.
Matrices based on native organic polymers, such as polysaccharides and especially agarose, are used for many chromatographic applications, especially in a derivatised form since the hydroxyl groups available on their surfaces render them easy to derivatise. Agarose particles can be cross-linked by addition of a chemical cross-linker, and they will become porous as a result of the gelling procedure. The nature of such particles is generally known as gelporous, and such gelporous particles are in general less advantageous in procedures where high pressures are used.
Matrices based on synthetic organic polymers, such as divinylbenzene (DVB) and styrene, on the other hand are tolerant to cleaning in place, but their aromatic groups can sometimes cause undesired effects in chromatographic methods. More specifically, if such a matrix is used to adsorb molecules that also comprise aromatic groups, an interaction between their respective π electrons will occur, resulting in peak tailing and long retention times. A disadvantage of DVB based matrices is that they often exhibit a large proportion of undesired micropores, which results in impaired mass transfer properties in chromatography. It has been hypothesised that these micropores, which are especially disadvantageous in RPC, are produced as a result of the rapid polymerisation of DVB monomers in suspension polymerisation. Another disadvantage of styrene based matrices appears in cases where it is desired to derivatise the surface of the particle to change its properties, since this kind of matrices exhibit a chemical inertness that in most cases necessitates several surface modification steps before the desired change can be achieved. A further disadvantage of organic polymer-based polymers such as styrene and DVB is that they are somewhat compressible at the high-pressure conditions that are used in high-performance separation methods.
In order to provide improvements to the above-discussed groups of matrix materials, newer alternatives have been presented. For example, in order to produce improved preparative ion exchange chromatography matrices of improved mechanical and chemical stability, Britsch et al (Recovery of Biological Products) have suggested a copolymer synthesised of a mixture of hydrophilic vinyl ether and a bifunctional acrylamide monomer. The resulting product is accordingly based on amide type cross-linking. However, as is well known, acrylamide requires care to be taken when handled to avoid negative health effects, which will result in a more cumbersome and inconvenient process. Furthermore, the hydrolysis sensitivity of amides in general may render this kind of matrices less advantageous for certain applications.
Ericsson et al (WO 95/13861) have disclosed a separation method, wherein hydrophilic vinyl ether polymers attached to a support are used as a matrix. More specifically, the polymers suggested therein comprise a poly(vinyl ether) chain comprising identical or different vinyl subunits, and the support is illustrated with agarose particles. The polymers are synthesised using cationic polymerisation.
Further, in order to avoid the disadvantages of alkyl-bonded silica matrices and polymer matrices in reversed phase liquid chromatography, Hirayama et al (Chromatographia Vol. 33, No 1/2, January 1992, 19–24) suggest suspension copolymerisation of alkylvinyl ether with triethylene glycol divinyl ether. Thus, in this method, the organic phase was comprised of two different kinds of reactive vinyl ether monomers, the mutual ratio of which was used to set a specific hydrophobicity of the resulting product.
U.S. Pat. No. 5,334,310 discloses a liquid chromatographic column that contains a separation medium in the form of a macroporous polymer plug, also known as a monolith. The polymerisation mixture from which the plug is prepared contains at least one polyvinyl monomer, a free radical generating initiator and a porogen. The so prepared plug contains small pores of diameters less than about 200 nm as well as large pores of diameters greater than about 600 nm. Such a large pore diameter range is advantageous for monoliths to allow a high flow rate, but clearly above the useful pore diameter ranges in polymer particles intended for chromatography.
EP 1 179 732 presents a solution as regards how to produce a polymer adsorbent that exhibits selected porosity and permeability characteristics. This has been achieved by a method, preferably a suspension polymerisation method, wherein selected mixed porogens in selected proportions relative to the monomer are used. The thereby produced adsorbent is due to its rigidity especially suitable for use as an RPC stationary phase at high-pressure conditions.
Accordingly, since the various groups of chromatographic matrix materials exhibit certain advantages and disadvantages, it is often the case that the matrix used is selected depending on the kind of target molecule, the purpose of the separation etc. Alternatively, two or more different chromatographic principles and/or different matrix materials are sometimes combined into sequence of chromatographic steps, often denoted polishing, capture etc. Thus, each separation principle can be viewed as one tool useful in a toolbox, where there is a constant need of new tools. Accordingly, despite the known matrix materials discussed above, there is still a need of novel methods to use as supplement, i.e. as further tools in a tool box.